TGF-β is a reacted protein that exists in at least three isoforms called TGF-β1, TGF-β2 and TGF-β3, and it controls cell proliferation and differentiation, wound healing, extracellular matrix production and immune-suppression. Other members of the transforming growth factor superfamily include activins, inhibins, bone morphogenetic proteins, growth and differentiation factors, and Müllerian inhibiting substance.
TGF-β1 transduces signals through two highly conserved single transmembrane serine/threonine kinases, the type I (ALK5) and type II TGF-β receptors. Upon ligand-induced oligomerization, the type II receptor hyperphosphorylates serine/threonine residues in the GS region of ALK5, which leads to the activation of ALK5 by creating a binding site for Smad proteins. The activated ALK5 in turn phosphorylates Smad2 and Smad3 proteins at the C-terminal SSXS-motif, thereby causing their dissociation from the receptor and heteromeric complex formation with Smad4. Smad complexes translocate to the nucleus, assemble with specific DNA-binding co-factors and co-modulators, to finally activate the transcription of extracellular matrix components and inhibitors of matrix-degrading proteases.
Activins transduce signals in a manner similar to TGF-β. Activins bind to serine/thereonine kinase, the activin type II receptor (ActRIIB), and the activated type II receptor hyperphosphorylates serine/threonine residues in the GS region of the ALK4. The activated ALK4 in turn phosphorylates Smad2 and Smad3. The consequent formation of a hetero-Smad complex with Smad4 results in the activin-induced regulation of gene transcription.
Numerous experimental animal studies have demonstrated that the glomerular expression of TGF-β is associated with fibrosis. Such studies include Thy-1 rat model of proliferative glomerulonephritis, anti-GBM glomerulonephritis in rabbits, and 5/6 nephrectomy rat model of focal segmental glomerulosclerosis, as has been recently reviewed (see, Bitzer, M. et al., Kidney Blood Press. Res. 21:1-12 (1998)). Neutralizing antibodies against TGF-β improve glomerular histology in Thy-1 nephritis model (see, Border, W. A. et al., Nature 346: 371-374 (1990)).
Hyperglycemic conditions promote the TGF-β mRNA and protein syntheses in both murine proximal tubule cells and human mesangial cells (see, Wahab, N. A. et al., Biochem. J. 316:985-992 (1996); Rocco, M. V. et al., Kidney Int. 41: 107-114 (1992)). Diabetic patients with an early kidney disease show increased accumulation of TGF-β mRNA and the expressed protein within the glomerulus (see, Yoshioka, K. et al., Lab. Invest. 68: 154-163 (1993)). Kidneys with chronic renal interstitial fibrosis exhibit thickened tubular basement membranes and an expanded interstitial compartment, with interstitial fibrosis characterized by an increase in collagens I, III, V, VII, and fibronectin (see, Eddy, A. A., J. Am. Soc. Nephrol. 7: 2495-2508 (1996)).
TGF-β gene expression and TGF-β protein production have been observed to increase in a variety of animal models of pulmonary fibrosis caused by bleomycin, silica, asbestos, and radiation (see, Phan, S. H. and Kunkel, S. L., Exp. Lung Res. 18: 29-43 (1992); Williams, A. O. et al., Am. J. Pathol. 142: 1831-1840 (1993); Rube, C. E. et al., Int. J. Radiat. Oncol. Biol. Phys. 47: 1033-1042 (2000)). Coincident increase in TGF-β1 protein and collagen gene expression in adjacent tissue slices from idiopathic pulmonary fibrosis is observed in human pulmonary fibrotic diseases (see, Broekelmann, T. J. et al., Proc. Natl. Acad. Sci. USA 88:6642-6646 (1991)). Increased TGF-β production has been observed for patients with sarcoidosis, pneumoconiosis, asbestosis, and radiation-induced fibrosis (see, Khalil, N. et al., Am. J. Respir. Cell. Mol. Biol. 14:131-138 (1996); Jagirdar, J. et al., Environ. Health Perspect. 105:1197-1203 (1997)). Anti-TGF-β antibodies and TGF-β-soluble receptors could partially inhibit fibrosis in bleomycin-induced lung fibrosis rodent models (see, Giri, S. N. et al., Thorax 48: 959-966 (1993); Wang, Q. et al., Thorax 54: 805-812 (1999)). Tobacco smoke has been implicated as one of the most important factors that cause small airway disorders, leading to chronic obstructive pulmonary disease (COPD) (see, Wright, J. M. et al., Am. Rev. Respir. Dis. 146: 240-262 (1992)). COPD is a slowly progressive and irreversible disorder characterized by the functional abnormality of airway obstruction. TGF-β has been hypothesized to be involved in airway remodeling of the chronic airway inflammatory disorders such as COPD (see, Takizawa, H. Int. J. Mol. Med. 1: 367-378 (1998); Ning, W. et al., Proc. Natl. Acad. Sci. USA 101:14895-14900 (2004)).
Hepatic stellate cells (HSC) are the major source of extracellular matrix proteins in hepatic fibrosis. Extracellular matrix production by activated hepatic stellate cells markedly increases by the action of TGF-β1 (see, Friedman, S. L., Prog. Liver Dis. 14: 101-130 (1996); Pietrangelo, A., Semin. Liver Dis. 16:13-30 (1996)). Transgenic mice that overexpress TGF-β1 in the liver develop hepatic fibrosis as well as extrahepatic pathologies such as renal fibrosis (see, Sanderson, N. et al., Proc. Natl. Acad. Sci. USA 92:2572-2576 (1995)).
TGF-β1 and its receptors are overexpressed in injured blood vessels and in fibroproliferative vascular lesions, leading to overproduction of extracellular matrix (see, Saltis, J. et al., Clin. Exp. Pharmacol. Physiol. 23: 193-200 (1996); McCaffrey, T. A. et al., J. Clin. Invest. 96: 2667-2675 (1995)).
Anti-TGF-β antibodies reduce scar formation with the improvement of the cytoarchitecture of the neodermis in rats (see, Shah, M., J. Cell. Sci. 108: 985-1002 (1995)), promote the healing of corneal wounds in rabbits (see, Moller-Pedersen, T., Curr Eye Res. 17:736-747 (1998)), and accelerate wound healing of gastric ulcers in rats (see, Ernst, H., Gut 39: 172-175 (1996)).
Radiation fibrosis is a frequent sequel of therapeutic or accidental radiation overexposure of normal human tissues. TGF-β1 plays a key role in the initiation, development, and persistence of radiation fibrosis, as has been recently reviewed (see, Martin, M. et al., Int. J. Radiat. Oncol. Biol. Phys. 47:277-290 (2000)).
Organ transplantation is often complicated by chronic rejection, which for some organs such as the kidney, becomes the major causes of graft loss. In human patients, chronic rejection of lung and kidney transplants is associated with increased expression of TGF-β within the tissue (see, El-Gamel, A. et al., Eur. J. Cardiothorac. Sung. 13: 424-430 (1998); Shihab, F. S. et al., J. Am. Soc. Nephrol. 6:286-294 (1995)).
TGF-β is implicated in peritoneal adhesions (see, Saed, G. M. et al., Wound Repair Regeneration 7: 504-510 (1999)). The peritoneal and sub-dermal fibrotic adhesions may be prevented by administering ALK5 and/or ALK4 inhibitors.
The tumor cells and the stromal cells within the tumors in late stages of various cancers generally overexpress TGF-β. This leads to stimulation of angiogenesis and cell motility, suppression of the immune system, and increased interaction of tumor cells with the extracellular matrix (see, Hojo, M. et al., Nature 397: 530-534 (1999)). Consequently, the tumor cells become more invasive and metastasize to other organs (see, Maehara, Y. et al., J. Clin. Oncol. 17: 607-614 (1999); Picon, A. et al., Cancer Epidemiol. Biomarkers Prev. 7:497-504 (1998)).
Plasminogen activator inhibitor-1 (PAI-1) is a major physiological inhibitor of both tissue-type plasminogen activator and urokinase-type plasminogen activator. Elevated levels of PAI-1 are associated with thrombosis and vascular disease, suggesting that high plasma PAI-1 may promote a hypercoagulable state by disrupting the natural balance between fibrinolysis and coagulation (see, Vaughan, D. E., J. Invest. Med. 46: 370-376 (1998)). It is known that TGF-β stimulates the expression of PAI-1 (see, Dennler, S. et al., EMBO J. 17: 3091-3100 (1998)). Accordingly, the inhibition of the production of PAI-1 with an inhibitor of the TGF-β signaling pathway would lead to novel fibrinolytic therapy.
Activin signaling and overexpression of activin are linked to pathological disorders that involve extracellular matrix accumulation and fibrosis (see, Matsuse, T. et al., Am. J. Respir Cell Mol. Biol. 13:17-24 (1995); Inoue, S. et al., Biochem. Biophys. Res. Comm. 205:441-448 (1994); Matsuse, T. et al., Am. J. Pathol. 148:707-713 (1996); De Bleser et al., Hepatology 26:905-912 (1997); Pawlowski, J. E., et al., J. Clin. Invest. 100:639-648 (1997); Sugiyama, M. et al., Gastroenterology 114:550-558 (1998); Munz, B. et al., EMBO J. 18:5205-5215 (1999)), inflammatory responses (see, Rosendahl, A. et al., Am. J. Respir Cell Mol. Biol. 25:60-68 (2001)), cachexia or wasting (see, Matzuk, M. M. et al., Proc. Natl. Acd. Sci. USA 91:8817-8821 (1994); Coerver, K. A. et al., Mol. Endocrinol. 10:534-543 (1996); Cipriano, S. C. et al., Endocrinology 141:2319-2327 (2000)), diseases or pathological responses in the central nervous system (see, Logan, A. et al., Eur J. Neurosci. 11:2367-2374 (1999); Logan, A. et al., Exp. Neurol. 159:504-510 (1999); Masliah, E. et al., Neurochem. Int. 39:393-400 (2001); De Groot, C. J. A. et al., J. Neuropathol. Exp. Neurol. 58:174-187 (1999); John, G. R. et al., Nat. Med. 8:1115-1121 (2002)) and hypertension (see, Dahly, A. J. et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 283: R757-767 (2002)). Studies have shown that TGF-β and activin can act together synergistically to induce extracellular matrix production (see, Sugiyama, M. et al., Gastroenterology 114:550-558 (1998)).
Therefore, it becomes evident that the inhibition of ALK5 and/or ALK4 phosphorylation of Smad2 and Smad3 by the compound of the present invention would be able to treat and prevent the above mentioned disorders related to said signaling pathways.
International Publication No. WO 00/61576, and U.S. Patent Application Publication No. US 2003/0149277 A1 disclose triarylimidazole derivatives and their use as ALK5 inhibitors. International Publication No. WO 01/62756 discloses pyridinylimidazole derivatives and their use as ALK5 inhibitors. International Publication No. WO 02/055077 discloses the use of imidazolyl cyclic acetal derivatives as ALK5 inhibitors. Also, International Publication No. WO 03/087304 discloses tri-substituted heteroaryls and their use as ALK5 and/or ALK4 inhibitors.
The present inventors have unexpectedly discovered that a class of 2-pyridyl substituted imidazoles function as potent and selective inhibitors of ALK5 and/or ALK4 receptors and therefore, have utility in the treatment and prevention of various diseases mediated by ALK5 and/or ALK4 receptors.